sditelberg's blog

We have focused our research on EMT thus far. The p53-miR-200c pathway, which is included in the family of miR200s that inhibit EMT, inhibits the activity of transcription factor ZEB when p53 is present in high levels. miR200s can also upregulate e-cadherin, possibly allowing for MET (https://www.nature.com/articles/cdd201142#s1). Additionally, reactive oxygen species (ROS) in high levels can lead to apoptosis, but in low levels can lead to cell growth. In cancer cells, antioxidant proteins also work to maintain ROS at a specific functional level (https://www.sciencedirect.com/science/article/pii/S0167488916302324). If used in treatment, specific levels of ROS would need to be determined to obtain the desired apoptotic effect. Additionally, levels of ROS in cancer cells would need to be increased to saturate these antioxidant proteins, or these proteins would need to be eliminated altogether. Perhaps ROS could somehow be incorporated into the interior of the exosome as well.

Efforts to halt EMT, induce MET, and initiate apoptosis can be included in phase 2 of treatment. These may be grounded in aptamer-regulated exosomal therapy, where aptamers can serve as binding partners for relevant transcription factors and signaling pathway elements. Binding partners for these aptamers have yet to be determined, however, they should be specific for pancreatic cancer cells. Various treatments can be included inside the exosomes, such as miR200c or other miRNAs in the 200 family, or an apoptosis-inducing molecule such as ONC201. To maximize efficiency of treatment, phases 1 and 2 can overlap slightly so all pancreatic cancer cells throughout the body may be recognized effectively during phase 2. This is especially true for the ever-changing tumor microenvironment and the premetastatic niche, which both still need to be researched more.

It is crucial to note that Group 6 did not have a 50 mM NaCl brain to image and therefore could not accurately compare data with the class average for this salt concentration to draw any significant conclusions. However, upon examining the system water and nanopure lines against the 50 mM NaCl and 100 mM NaCl lines throughout the brain for the class, there is no clear trend of the two salt lines with increased cell counts, which does not support the hypothesis. Although the 50 mM NaCl line had higher cell counts than the system water and nanopure in the hypothalamus, the 100 mM NaCl line had similar cell numbers to the two lines without salt. A similar result is seen in the class data of the lateral recess: 100 mM NaCl had slightly higher counts than the system water and nanopure lines, but 50 mM NaCl had lower counts than the two lines without salt. Perhaps this Group 6 100 mM NaCl brain is a true outlier in cell proliferation numbers due to experimental treatment, or perhaps differences may be explained through variation in cell counting and techniques. The members of Group 6 obtained this brain from a separate experimental vial given by a TA and did not dissect it themselves, which may have contributed to the variation in cell numbers seen.

The antigen CD44 is expressed on the surface of pancreatic cancer stem cells (PCSCs) and modulates the cytoskeleton via the linker protein ezrin, which is more active and present in higher levels in these stem cells than in differentiated tumor cells (Penchev et al. 2019). Elimination of ezrin as well as targeting via a small molecule inhibitor has been found to decrease self-renewal, clonogenic growth, and migration in vitro as well as tumor initiation in vivo (Penchev et al. 2019). Natural compounds and phytochemicals such as curcumin, resveratrol, tea polyphenol EGCG (epigallocatechin-3-gallate), crocetinic acid, sulforaphane, genistein, indole-3-carbinol, vitamin E δ-tocotrienol, plumbagin, quercetin, triptolide, licofelene, and quinomycin have also been shown to inhibit PCSCs as well as their signaling pathways (Subramaniam et al. 2018). These treatments to inhibit self-renewal may be useful in phase 1 of our treatment plan, but a method to induce differentiation back into pancreatic cancer cells has yet to be researched. The premetastatic niche may also be able to be targeted in phase 1 of treatment, but effective targets have yet to be researched as well.

Regarding the carnivorans, the character #6, tail, has two character states. In this phylogenetic analysis, an elongated tail is the ancestral character state (scored with a 0) and a short tail is the derived character state (scored with a 1). In phylogeny A, a short tail is hypothesized to have evolved after the split between otters and the group of bears, sea lions, walrus, and seals. This proposes that a short tail is the synapomorphy for the monophyletic group of bears, sea lions, walrus, and seals. In phylogeny B, a short tail is hypothesized to have evolved twice. This is an example of homoplasy. For example, a short tail here is a derived trait for the seals, but it is also a shared derived trait for bears, sea lions, and walrus. However, there are a few separate divergences between seals and this group, and the common ancestor is hypothesized to have an elongated tail. In phylogeny C, a short tail is hypothesized to have evolved twice as well, but then lost in one lineage branch. For example, a short tail is a derived trait for the bears, but it also initially evolved as a shared derived trait for the sea lions, walrus, seals, civets, hyenas, and cats taxa. Cats, hyenas, and civets then lost this short tail trait. This is an example of an evolutionary reversal. In phylogeny D, a short tail evolved once in the lineage to include its monophyletic group branching from seals to dogs, but then this trait was lost later in the phylogeny in otters, raccoons, and dogs. This is another example of an evolutionary reversal. Based on this trait and the parsimony principle, phylogeny A is the most likely. The parsimony principle guides us to the evolutionary tree with the fewest character-state changes, and this is the one usually regarded as the best. In phylogeny A, the tail trait only evolved once in the lineage and was not lost at any point.

To improve the quality and consistency of the experiment and cell counts across the class, the exact regions of the brain to be counted (e.g. telencephalon, posterior recess, etc.) could be encircled first. Implementing a standardized system for identifying regions of the brain is crucial to both accuracy and precision of experimental technique. This has already been partially started, but could be expanded upon and continued. Level of consistency in the brains obtained could also be improved. Because of low fish survival, salt-treated brains were obtained from a separate experiment, which may have led to differences in cell numbers, affecting the accuracy of the results. Perhaps less strong salt concentrations can be used when allowing embryos to grow to improve survivability. Salt-treated brains from the same experiment as the system H2O and nanopure brains can then be dissected and counted. Any group with extra salt-treated brains can give them to groups that do not have as many. Perhaps this may improve the accuracy of cell counting on the whole. Fixing protocol of the zebrafish larvae could also be changed to improve ease of dissection, allowing more time to run the experiment. After break, weaker salt concentrations could be experimented with and an improved protocol could be run, or a different variable could be tested. pH could be an interesting variable to explore with the zebrafish. Could slight changes in pH alter cell proliferation rates in the brain, and if so, would these changes occur in the same regions?

It is crucial to note that Group 6 did not have a 50 mM NaCl brain to image and therefore could not accurately compare data with the class average for this salt concentration to draw any conclusions that may or may not have matched the prediction and supported the hypothesis. However, upon examining the system H2O and nanopure lines against the 50 mM NaCl and 100 mM NaCl lines throughout the brain for the class, there is no clear trend of the two salt lines with increased cell counts. This does not match the prediction and support the hypothesis. Although 50 mM NaCl had higher counts than the system H2O and nanopure in the hypothalamus, 100 mM NaCl had similar cell numbers to the two lines without salt. A similar result is seen in the class data of the lateral recess: 100 mM NaCl had slightly higher counts than the system H2O and nanopure lines, but 50 mM NaCl had lower counts than the two lines without salt. Perhaps this Group 6 100 mM NaCl brain is a true outlier in cell proliferation numbers due to experimental treatment, or perhaps differences may be explained through variation in cell counting and techniques. The members of Group 6 obtained this brain from a separate experimental vial given by a TA and did not dissect it themselves, which may have contributed to the variation in cell numbers seen.

Although statistical analyses have not yet been performed on these data, the Group 6 100 mM NaCl brain had higher average cell counts than those of the class in most regions of the brain, most noticeably in the hypothalamus and the telencephalon. This result alone matches the prediction that by altering salt conditions, cell proliferation rates will increase. This result alone also supports the hypothesis as well. It is crucial to note that Group 6 did not have a 50 mM NaCl brain to image and therefore could not accurately compare data with the class average for this salt concentration to draw any conclusions that may or may not have matched the prediction and supported the hypothesis. The system H2O and nanopure brains of Group 6 had slightly lower average cell counts than those of the class. It is important to note that during imaging of the nanopure brain, the telencephalon was cut off, therefore no accurate cell count could be obtained. These slight differences in cell counts for the system H2O and nanopure lines could be due to variation in counting as the greatest difference throughout individual regions of the brain is approximately 20 cells, exhibited in the lateral recess. For the system and nanopure lines, there is a difference of approximately 35 cells in the hypothalamus, yet this is a summation of the posterior and lateral recesses, which have differences of approximately 15 and 20 cells between Group 6 and the class averages, respectively. With other individual regions of the brain exhibiting these slight differences in counts as well, it is likely that the system H2O and nanopure lines vary due to counting or respective techniques.

Depending on their native environment, different species of fish respond to osmotic changes in different ways. Some fish are able to live in multiple salinity concentrations and are known as euryhaline, while others are only able to survive in one and are known as stenohaline. Both stenohaline and euryhaline fish tightly regulate internal concentrations of salt and water by tending to excrete more of the substance present in their environment: marine fish excrete large amounts of salt and little amounts of water, whereas freshwater fish excrete large amounts of water and little amounts of salt (Karlstrom 2019). This ionocytic regulation itself is regulated by the hypothalamic-pituitary axis in the brain, which can be further investigated through the zebrafish, a freshwater model organism. It is hypothesized that as a response to changing osmotic demands, the zebrafish hypothalamus alters its dopamine hormone output in the short-term to the pituitary (which in turn regulates prolactin and ionocytes) and in the long-term alters cell proliferation rates to establish new regulatory populations of dopaminergic neurons. If zebrafish are exposed to altered salt concentrations in their environment, cell proliferation rates of dopaminergic neurons in the hypothalamus will increase as a long-term response to this new, stressful osmotic demand.